Alcohol oxidase (AO) is an enzyme that catalyzes the oxidation of C.sub.1 -C.sub.4 straight chain alcohols to corresponding aldehydes and thereby produces hydrogen peroxide. The enzyme is produced in methanol-utilizing yeasts such as P. pastoris, Hanesenula polymorpha, and Candida boidinii, when methanol is present in the growth media. These yeasts assimilate methanol by oxidizing methanol to formaldehyde and hydrogen peroxide. Since the hydrogen peroxide formed is extremely toxic to these yeasts and, it is normally immediately converted to molecular oxygen and water by another enzyme, catalase.
To be useful in washing, bleaching and other aseptic processes, there is a need to produce AO without catalase attached. The AO-catalase enzyme complex produced by methylotrophic yeasts can be isolated from cell-free extracts followed by removal of the catalase so that the catalase-free AO is suitable for use, for example, in a detergent composition to produce hydrogen peroxide upon reaction with a substrate. The process is, however, prohibitively expensive when used to produce catalase-free AO in bulk quantity. Additionally, any residual catalase will still convert hydrogen peroxide to oxygen and water.
Accordingly, it is advantageous to be able to produce AO from yeasts while avoid producing catalase. One such process for producing catalase-free AO has been disclosed. For example, European Patent Application 242,007 discloses the production of catalase-free methanol oxidase in catalase-negative mutants of Hansenula polymorpha grown in a nutritive medium suitable for the yeasts in the presence of another source of carbon such as glucose, in which methanol induces the expression of the methanol oxidase gene and is also used as a substrate for the oxidase, while the toxic effects of the hydrogen peroxide produced are circumvented by using a suitable mixing ratio of methanol to other source of carbon. However, such Hansenula polymorpha catalase-negative mutants yield only 49% of methanol oxidase with respect to the wild-type strain cultured on methanol.
There is, therefore, a need for a yeast that can produce large quantity of catalase-free AO.
It is therefore an object of the invention to provide a novel Pichia pastoris which will produce large quantities of catalase-free AO.
It is also an object of the invention to provide a process to produce the catalase-free AO in high yield by cultivating catalyse-free strain of P. pastoris by co-substrate fermentation.
Other aspects, objects, and the several advantages of this invention will be apparent from the following specification and claims.